The present invention relates to an optical sensor unit and to a procedure for accurate and ultrasensitive detection of chemical or biochemical analytes. An optical sensor unit is described combining several unique parts inching a laser light source, an optical detection module, and an integrated optical transducer chip. The optical transducer chip consists of a disposable carrier substrate coated with a waveguiding layer onto which sensing biomolecules are bonded in pattern fashion, said sensing biomolecules being capable of interacting with chemicals or bio-molecules of an analyte solution to induce changes in the effective refractive index.
More particularly, the invention relates to the detection of prions and prion binding molecules in pico- to femto-molar concentrations with the help of antibodies, preferably monoclonal, allowing specific detection of disease-specific prion proteins on surfaces engineered for low non specific binding of bioconstituents. Conversely, the biosensor is applicable to identify ligands to prion proteins if instead of the monoclonal antibodies, recombinant or highly purified PrP is bonded to the optical chip by a one-step procedure, which can be performed even by those not skilled in the art.
More generally, the sensor and the procedure according to the invention facilitates ligand screening of chemical and biochemical libraries.
As the detection of prions and prion binding molecules is at this time of upmost importance for diagnosing and treating prion related diseases, this aspect will serve as guideline for the description of the invention. For a better understanding the abbreviations used hereinbefore and hereinafter are the following:
The references to the prior art reported hereinafter will be termed by the name of the first author and the publication year as mentioned below:
Basler, K., Oesch, B., Scott, M., Westaway, D., Walchli, M., Groth, D. F., Mc Kinley, M. P, Prusiner, S. B., and Weissmann, C. (1986). Scrapie and cellular PrP isoforms are encoded by the same chromosomal gene. Cell 46, 417-428.
Borchelt, D. R., Scott, M., Taraboulos, A., Stahl, N., and Prusiner, S. B. (1990). Scrapie and cellular prion proteins differ in their kinetics of synthesis and topology in cultured cells. J. Cell Biol. 110, 743-752.
Bueler, H., Fischer, M., Lang, Y., Bluethmann, H., Lipp, H. P., DeArmond, S. J., Prusiner, S. B., Aguet, M., and Weissmann, C. (1992). Normal development and behaviour of mice lacking the neuronal cellsurface PrP protein. Nature 356, 577-582.
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The disease-specific forms of the prion protein (PrPSc, PrPBSE) are part of the infectious particle causing transmissible neurodegenerative diseases like scrapie in sheep, bovine spongiform encephalopathy in cattle or Creutzfeldt-Jakob disease in humans. PrPSc as well as PrPBSE differ from the normal cellular prion protein (PrPc) by their relative protease resistance (Oesch, 1985). The molecular changes leading to this difference in physicochemical properties are unknown. Protease-resistant PrPSc and infectivity to date have not been separated, leading to the proposal that the infectious particle would be composed of specifically altered PrP molecules. The primary amino acid sequence of PrPSc is identical to that predicted from its cDNA sequence or genomic nucleic acid sequence (Stahl, 1993) and the infectious particle does not encode an altered PrP gene (Oesch, 1985). In cell culture, PrPc is converted into PrPSc posttranslationally (Borchelt, 1990). However, no differences in covalent modifications of PrPSc and PrPc were observed by mass spectrometry (Stahl, 1993). The lack of a molecular explanation for the observed differences between PrPSc and PrPc led to the proposal that the PrP isoforms differ in their conformation (Basler,1986). The relative protease resistance of PrPSc, e.g. PrPc being fragmented by proteinase K and PrPSc being partially resistant to the action of proteinase K, is at this time the usual way to distinguish the two forms of PrP (Serban, 1990).
PrPSc is currently detected by Western or dot blotting. After protease digestion, the molecular weight of PrPSc is reduced from 33-35 kDa to 27-30 kDa (PrP 27-30). This characteristic change in molecular weight is detected by Western blotting and serves as a hallmark of PrPSc. As an alternative, PrP 27-30 can be detected on dot blots (Oesch, 1994; Korth, 1997). Native PrP 27-30 is not recognized by antibodies, whereas the denatured form of the protein interacts with antibodies. (Serban, 1990). Circumstantial evidence indicates that the epitopes in PrPc and PrPSc differ in accessibility. Previously, it has been attempted without success to generate antibodies which specifically recognize PrPSc, thus limiting the immunological detection methods to procedures including denaturation of samples prior to detection. However, (Korth, 1997) described monoclonal antibodies (see below) that do allow detection of native PrPSc and, specifically, PrPBSE.
A receptor for PrP transducing biological effects of PrP has not been found yet. Until now, various proteins have been shown to interact with PrP: glial fibrillary acidic protein (GFAP); (Oesch, 1990), bcl-2 (Kurschner, 1995). All of these proteins are intracellular proteins which contrasts with the cell surface location of PrP. Even though there may be a role for these PrP binding proteins, in particular bcl-2, in the neurotoxic effects of PrPSc it is postulated that a cell surface protein should exist which binds to PrP. Despite the scientific investigations summarized above, there is still a need for a detection system which impairs as little as possible the conformation of the molecules or biomolecules to be detected.
Indeed, known analyte detection techniques have their limitations in terms of sensitivity and quantification, the procedure involving several steps, the necessity of detection of labelled components and the overall duration of the test procedure.
With the present invention, these shortcomings are overcome by the use of biosensors which allow the registration of molecular interactions at the surface of integrated optical chips. The basic principle of these biosensors is to measure the change in the effective refractive index for the guided waves on an optical chip when a ligand interacts non-covalently with molecules bonded to the surface of the respective chip. The specificity of the interaction is ascertained by the use of molecules such as antibodies immobilized on the surface of integrated optical chips. The described PRPBSE biosensor detection system is based on an optical transducer in combination with miniature integrated optical sensor devices. Optical waveguide sensing provides the features of exceptional high sensitivity and label free detection of analytes within the evanescent field of incoupled laser light. Requirements for high selectivity, reduction of non-specific binding and multiple sensing on a single waveguide surface are accomplished by appropriate sensor surface bioengineering combined with multidomain photobonding. The use of multidomain analysis on integrated optical chips dramatically simplifies the instrumental features of the detection system. Multidomain analysis on a single chip enables integrated calibration and referencing. The described prion detection system in combination with unique monoclonal antibodies is used to detect PRPBSE with high sensitivity and high speed. It allows to screen large numbers of samples.
With the same detection system, on the other hand, biomolecules interacting with PrP can be identified. For this purpose, instead of covalently bonding monoclonal antibodies to PrP, rb PRP or highly purified native PrP, PrPc or PrPSc is immobilized on the surface of the optical chip. Since. applied detection principles do not require labelling of a probe, it is exquisitely suited to search for (an) elusive xe2x80x9cPrP receptor(s)xe2x80x9d which is (are) currently unknown but postulated to be essentially involved in the modulation of PrP function as well as in the infection of cells by prions.
The described biosensor system further suits the screening of chemical libraries in search for potential ligands which interact with molecules bonded to optical chips. For example, PrP bonded on an optical chip allows one to screen for chemical ligands capable of preventing neurotoxic and infectious properties of prions.
It is the object of the present invention to overcome the drawbacks and failures of prior art like complex multistep procedures, limited sensitivity, necessity of label-based analyte detection and necessity of sophisticated equipment. These limitations are addressed in the present invention, which provides an accurate and ultrasensitive detection system for biological ligands, especially prion proteins and proteins which interact with prion proteins, to be used in the diagnosis of prion diseases, or any other biological state involving detection of molecules in biological fluids or tissue homogenates and requiring high sensitivity.
The present invention describes a novel sensor system which is able to detect and quantify pico- to femto-molar concentrations of chemicals or bio- molecules in analytes of biological origin in real fluids. Analytical limitations of hitherto systems are overcome by combining the exceptional sensitivity of a new design for simplified integrated optical detection on disposable sensor chips with the intrinsic specificity of sensing bioconstituents and photobonding and biopatten technologies enabling both array detection and integrated calibration. Teachings include the design of the biosensor system and its essential components, the design and fabrication of multi-array biosensor chips and the preparation and characteristics of high-affinity disease-specific bioconstituents. General use of the biosensor system for on-site application is exemplified with the detection of PrPBSEin homogenates of brain and the detection of molecular components participating in prion related diseases, e.g. identification of PrPBSE binding biomolecules.
The present invention concerns the layout and description of the modular system components of the analytical instrument. In its final form, the instrument is portable and at least consists of a laser light source, an optical detection module and an integrated optical transducer chip. This instrument can be extended with an effector system, such as a fluid handling system for allowing the selection of individual samples, solvents or washing solutions, and with an appropriate computer-like component for data storage, processing and presentation.
The surface of the integrated optical transducer chip can be grafted with lipids, forming lipid bilayer surfaces, serving as a membrane mimetic systems for the integration of membrane receptor proteins.
Sensor pad illumination is achieved by directing the radiation from sources such as laser diodes in the red or near infra-red wavelength region (e.g. Sharp LT026MDO, xcexxcx9c785 nm), either directly or via optical fibers. The optical output signal is detected by one- or two-dimensional detector devices, such as charge coupled devices (CCDs), photodiode arrays (PDAs), such as Hamamatsu S5463, or position sensitive devices (PSDs). The signal is evaluated and stored in digitized form and prepared for transmission to the computer component, e.g. RS232 interface.
In a preferred embodiment, the optical transducer chip consists of a high refractive index dielectric waveguiding film (e.g. TiO2, Si3N4, mixtures of TiO2 and Si3N4, Ta2O5, ZrO2, HfO2, Nb2O5), coated on a previously structured substrate (e.g. polycarbonate or polystyrene). The substrate is structured with (chirped) grating pads by means of procedures, as e.g. hot-embossing or injection moulding. The grating pads fulfill the tasks of coupling the light into the waveguiding film, of on-chip signal generation and of coupling the light out of the waveguide and directing it to the detector. A typical size of one sensor pad is in the order of a few mm2. Arrays of sensor pads can be accomplished by placing several pads next to each other. Using sensor pad arrays is advantageous for chemical multicomponent analysis and on-chip referencing. Preferred shapes of the biosensor chip are rectangular (card) or circular (disc). If the transducer is in the form of a disc, the disc is rotated on a central axis and contains optical on-chip elements for speed control and pad-specific information (e.g. position, sensing layer etc.). In the disc format, the sensing pads are arranged in concentric circles or in spiral arrangements. Either each pad is addressed consecutively, or several, or all pads can be interrogated in parallel by means of single or multiple beam illumination, e.g. by means of light source and detector arrays.
The sample holder consists either of an open cell allowing manual sample application, or it consists of a closed cell with tubings (e.g. Teflon, Peek) and a suitable liquid handling system.
The present invention concerns further a process for chip surface engineering including bonding of biomolecules, such as PrP, monoclonal or polyclonal antibodies, mixtures of monoclonal antibodies or other proteins which recognize specific epitopes on the target proteins (e.g. PrP). In a preferred embodiment, these proteins or antibodies are covalently immobilized on the waveguiding surfaces by means of photolinker polymer mediated photobonding or by oriented immobilization of analyte recognizing receptor molecules, genetically engineered receptor molecules or fragments of analyte recognizing immunoreagents through surface grafting with e.g. bifunctional photoactivatable maleimides. As an alternative, antibodies or other proteins are immobilized on the chip surface by absorption.
In addition to the immobilization methods discussed above, the layer of sensing bimolecules can be immobilized on the surface of a wave guiding layer by Langmuir-Blodgett techniques.
The present invention concerns further the process of engineering of the optical transducer surface with sensing biomolecules appropriate for analyte detection. Using mask-free or mask-assisted patterning procedures, the individual pads on the optical transducer surface are individually functionalized with selected molecules and varying compositions of biomolecules. For referencing, given sets of pads are modified with non-specific interacting analogs of the specific analyte-binding molecules, and on chip referencing is attained by sets of pads which provide increasing amounts of specific analyte binding molecules. Differential analyte binding kinetic data recorded from these calibration pads allow on-chip analyte calibration with reference to established binding constants.
The present invention concerns further a process, in which non-specific binding of bioconstituents present in the analyte fluid is suppressed by surface coating with polyethylene glycol, antibodies which do not interact with the ligand, polycarbohydrates, carbohydrates presenting low molecular weight photolinkers, or mixtures of the components mentioned.
The invention concerns further a procedure for verifying the proper functioning of the integrated optical transducer in situ, before and after each measurement by varying the incidence and/or the wavelength of the light.
The present invention concerns further a process in which the output signal light of a first optical sensor unit can be used to regulate, optionally by means of a computer, a second effector system and thus allowing the automation of biochemical reactions involving many single sequential or parallel steps.
The present invention concerns further a combination of the analytical detection system, the optical biosensor module bonded with monoclonal antibodies to PrP able to detect PrP in a fluid guided to the optical sensor unit.
The present invention concerns further a combination of the analytical detection system, the optical biosensor module with photobonded or oriented immobilized PrP able to detect biomolecules which non-covalently interact with said immobilized PrP.
The present invention concerns further a process for the identification and medical use of those biomolecules which can be detected and identified after non-covalent binding to surface-bonded PrP if said biomolecules inhibit the propagation of PrP destabilization or reverse PrP unfolding.